Gene expression in stage-specific populations of Rickettsia prowazekii

普瓦泽基立克次体特定阶段群体的基因表达

• 批准号: 8606398
• 负责人: DAVID WOOD
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-18 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8606398
• 关键词: Address; Aerosols; Affect; Arthropods; Bacteria; Bacterial Infections; Cell Line; Cell Separation; Cells; Characteristics; Codon Nucleotides; Coupled; Cytolysis; Cytosol; Data; Disease; Environment; Epidemic; Epidemic Louse-Borne Typhus; Evaluation; Exhibits; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; Gene Expression; Gene Expression Profile; Genes; Goals; Growth; Harvest; Heterogeneity; Human; Infection; Lice; Membrane; Molecular; Pathogenicity; Play; Population; Protein Array; Proteins; Proteomics; Public Health; Quantitative Microscopy; Reporting; Research Design; Rickettsia; Rickettsia prowazekii; Safety; Severity of illness; Staging; System; Technology; Time; Typhus; Vaccines; Virulence; Virulent; War; base; combat; design; environmental change; human disease; intracellular parasitism; mortality; novel; novel therapeutics; pathogen; public health relevance; transmission process; vector

DESCRIPTION (provided by applicant): Epidemic typhus, one of the most serious bacterial diseases affecting humans, is caused by the louseborne pathogen, Rickettsia prowazekii. Due to the high mortality rate of this disease, the lack of an effective vaccine, and the possibility o aerosol dissemination, R. prowazekii is designated as a select agent and has the potential to pose a severe threat to public health and safety. R. prowazekii is an obligate intracellular, parasitic bacterium that grows only within the cytosol of the host cell, unbounded by a vacuolar membrane. This ability to exploit the intracellular environment and cause human disease provides the basis for our studies designed to elucidate the mechanisms underlying obligate intracellular growth and R. prowazekii virulence. This proposal addresses the identification of critical stage-specific genes that are regulated as a rickettsial infection proceeds from a few rickettsiae per cell to hundreds per cell. A major hurdle in this field is the heterogeneity in the bacterial load within an infected population and the inability to generate synchronous rickettsial cultures. To tackle this problem, we propose to use a fluorescence activated cell sorting (FACS) approach to separate R. prowazekii-infected host cells, based on bacterial load, into distinct stage-specific populations. Using genetically-modified GFP-expressing R. prowazekii, we have demonstrated that FACS can be used to separate R. prowazekii-infected host cells into distinct populations harboring uniform numbers of rickettsiae per cell (population gating). We hypothesize that FACS-based separation of rickettsiae-infected cells will facilitate identification of rickettsial genes that are regulated at specific growth stages and that play key roles in optimizing the growth, virulence, and subsequent spread of this intracellular pathogen. We will address this hypothesis through the following specific aims: In Aim 1 we will identify and separate R. prowazekii-infected host cells into distinct populations according to bacterial load. Rickettsial numbers in isolated populations will be evaluated by microscopy and quantitative PCR. Optimization will include the evaluation of a codon-adapted fluorescent protein. We will evaluate different host cell lines (e.g. vertebrate and arthropod) and a virulent rickettsial strain. The effect of initial host cell infection levels and harvest times wll be examined. In Aim 2 we will identify, at both the transcriptional (gene arrays) and protein level (differential proteomics), genes that are differentially expressed as the rickettsiae transition frm exponential growth to lysis stage. Completion of this project will significantly advance the field, providing an accurate assessment of gene expression at defined stages in the R. prowazekii intracellular growth cycle. This data will lay a foundation for further delineation of molecular mechanisms underlying the infection, intracellular growth, and virulence of R. prowazekii and may reveal new targets for design of novel therapeutics to combat this resourceful and dangerous pathogen.

描述（由申请人提供）：流行性斑疹伤寒是影响人类的最严重的细菌性疾病之一，由虱传病原体普瓦泽基立克次体引起。由于该疾病死亡率高、缺乏有效的疫苗以及气溶胶传播的可能性，普罗瓦泽基立克氏菌被指定为选择性病原体，有可能对公众健康和安全构成严重威胁。普罗瓦泽基立克氏菌是一种专性细胞内寄生细菌，仅在宿主细胞的胞浆内生长，不受液泡膜的限制。这种利用细胞内环境并引起人类疾病的能力为我们的研究提供了基础，这些研究旨在阐明专性细胞内生长和普罗瓦泽基菌毒力的机制。该提案致力于鉴定关键阶段特异性基因，这些基因在立克次体感染从每个细胞的几个立克次体发展到每个细胞的数百个立克次体时受到调节。该领域的一个主要障碍是信息的异质性 感染人群中的细菌负荷以及无法产生同步立克次体培养物。为了解决这个问题，我们建议使用荧光激活细胞分选（FACS）方法根据细菌负荷将普罗瓦泽基立克氏菌感染的宿主细胞分离成不同阶段特定的群体。使用基因修饰的表达 GFP 的普罗瓦泽基立克次体，我们证明了 FACS 可用于将普罗瓦泽基立克次体感染的宿主细胞分离成每个细胞含有均匀数量的立克次体的不同群体（群体门控）。我们假设基于 FACS 的立克次体感染细胞分离将有助于鉴定立克次体基因，这些基因在特定生长阶段受到调节，并且在优化这种细胞内病原体的生长、毒力和随后的传播中发挥关键作用。我们将通过以下具体目标来解决这一假设：在目标 1 中，我们将根据细菌负荷识别普罗瓦泽基立克氏菌感染的宿主细胞并将其分成不同的群体。分离群体中的立克次体数量将通过显微镜和定量 PCR 进行评估。优化将包括评估密码子适应荧光蛋白。我们将评估不同的宿主细胞系（例如脊椎动物和节肢动物）和 一种强毒立克次体菌株。将检查初始宿主细胞感染水平和收获时间的影响。在目标 2 中，我们将在转录（基因阵列）和蛋白质水平（差异蛋白质组学）上鉴定当立克次体从指数生长过渡到裂解阶段时差异表达的基因。该项目的完成将显着推进该领域的发展， 提供对 R. prowazekii 细胞内生长周期的特定阶段的基因表达的准确评估。这些数据将为进一步描述普罗瓦泽基菌感染、细胞内生长和毒力的分子机制奠定基础，并可能揭示设计新疗法的新目标，以对抗这种资源丰富且危险的病原体。